The main goal of the present study is to explore the role of mono-ubiquitination of ROMK in the regulation of ROMK channel activity in response to stimulation of protein kinase A (PKA) and protein tyrosine kinase (PTK). ROMK is an inwardly-rectifying K channel and responsible for K recycling across the apical membrane of the thick ascending limb (TAL) and K secretion in the cortical collecting duct (CCD). The ROMK channel has three putative PKA-phosphorylation sites and one PTK-phosphorylation site. Stimulation of PKA-induced phosphorylation of ROMK channel augments the channel activity whereas increases in tyrosine-phosphorylation decrease the ROMK channel activity. Our preliminary data have shown that ROMK1 can be mono-ubiquitinated. Two lines of evidence indicate that lysine residue 22 (Lys22) is an ubiquiting binding site of ROMK1: 1) mutation of Lys22 to arginine increases the channel activity and surface expression of ROMK1;2) ubiquitination of ROMK1 is absent in oocytes injected with R1K22R. Also, stimulation of PKA decreases the mono-ubiquitination of ROMK whereas low K intake, which has been shown to increase PTK activity, enhances the ubiquitination of ROMK. We will test the hypothesis that the mono-ubiquitination of ROMK1 is involved in the regulation of ROMK channel number in the cell membrane and that the ubiquitination process of ROMK1 is modulated by PKA and PTK. Specific Aim 1 To determine the role of mono-ubiquitination of ROMK in mediating the internalization and to examine whether Nedd4, a ubiquitin ligase E3, is involved in mediating mono-ubiquitination of ROMK. Specific Aim 2 To examine whether mono-ubiquitination of ROMK channels is regulated by PKA and PTK. Specific aim 3 To explore the role of CD63 in mediating ROMK1 tyrosine phosphorylation and mono-ubiquitination.